Phytochemical Constituents, Antimicrobial Properties and Bioactivity of Marine Red Seaweed (Kappaphycus alvarezii) and Seagrass (Cymodocea serrulata)

The present work was performed to evaluate the levels of phytochemical constituents and the antioxidant and antibacterial properties of marine red seaweed (Kappaphycus alvarezii) and seagrass (Cymodocea serrulata). Quantitative phytochemical analysis, antioxidant activity and antimicrobial activity against five potential pathogenic bacteria was investigated. In each case, we found the presence of flavonoids, tannins, phenolic compounds, glycosides, steroids, carbohydrates and ashes. Alkaloids were only found in K. alvarezii, though they were not found in C. serrulata. The antimicrobial properties of both K. alvarezii and C. serrulata chloroform extracts were found to be antagonistically effective against the Gram-positive bacteria Bacillus subtilis and the Gram-negative bacteria Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi and Klebsiella pneumoniae. GC-MS analysis revealed the presence of 94 bioactive compounds in K. alvarezii and 104 bioactive compounds in C. serrulata, including phenol, decane, dodecane, hexadecane, vanillin, heptadecane, diphenylamine, benzophenone, octadecanoic acid, dotriaconate, benzene, phytol, butanoic acid and 2-hydroxyl-ethyl ether, which all played important roles in antioxidant and antibacterial activities. Thus, in view of the results, both K. alvarezii and C. serrulata could be considered to be sources of ingredients with appreciable nutritional and medicinal value.


Introduction
Marine organisms are valuable sources of bioactive compounds used by both the food and pharmaceutical industries. Bioactive compounds can be obtained from a wide range of marine foods. Nowadays, more than 36,000 compounds with potential effects on human health have been isolated from marine organisms [1]. Significantly, such bioactive compounds can minimize chronic non-communicable disease risk by reducing the onset of inflammation and oxidation [2]. In recent years, seaweeds have been reported to be an important source of bioactive compounds [3,4]. Another marine organism that contains a large variety of bioactive compound is seagrass, which, although it is less used than seaweeds, has been used in food and medicine by populations of coastal region [5].
Seaweeds are a group of autotrophic, halophytic and complex communities that live in marine environments and have the potential to be used as renewable resources [6,7]. Biologically, they are classified as either Phaeophyta (brown algae), Rhodophyta (red algae) leaf scars, as well as triangular and flat leaf sheath fibrous roots on the shoot and serrated leaf tips [10].
Although the composition of K. alvarezii was previously reported as being of the same origin, while the phytochemical composition of C. serrulata was also previously reported, it should be considered that the compositions of properties of both seaweed and seagrasses widely vary depending on a large variety of factors [4,5]. Thus, in a scenario in which important factors, such as ocean acidity, salinity or temperature, are changing [21], it is important to have updated data regarding seaweed and seagrasses composition and bioactivities. Hence, the aim of the present study was to evaluate the phytochemical constituents, antioxidant activity and antibacterial activity present in seaweeds and seagrass. The chemical compounds present in both K. alvarezii and C. serrulata were also determined.

Collection, Identification and Processing
The red seaweed K. alvarezii and the seagrass C. serrulata were obtained from Thondi coastal waters (Latitude: 9°44"N and Longitude: 79°00"E) in Palk Bay, India. Freshly collected seaweed (K. alvarezii) and seagrass (C. serrulata) ( Figure 1) were cleaned thoroughly in seawater and transported to the laboratory in a portable cooler that was protected from sunlight within 1 h of collection. The epiphytes, necrotic parts, muds, dust and other debris were removed via thorough washing with fresh water and double-distilled water. Next, they were shade-dried in an oven (Memmert, Roentgen, Germany) at 25 ± 2 °C for one week, ground into fine powder and stored at room temperature in an airtight container (Tarsons, Chennai, India) until analysis. The collected seaweed and seagrass were identified according to those standards established in the standard manual of Rao [22].

Preparation of Extracts
The seaweed and seagrass extracts were made by adding 5 g of dried seaweed or seagrass powder into 50 mL of three different solvents-chloroform, ethanol and distilled water-in a conical flask and placing the solution in a dark bottle in light agitation (AGI-MATIC-N, J.P. SELECTA S. A, Barcelona, Spain) for 7 days. After that, the extracts were filtered through Whatman No. 1 filter papers and sterile cotton wools, and the supernatants were stored at 4 °C for future use [23,24].

Determination of Alkaloids
The alkaloid contents of K. alvarezii and C. serrulata were determined via the method proposed by Hikino et al. [25]. Next, 1 mL of test extract phosphate buffer (5 mL, pH 4.7) was added to 5 mL of bromocresol green solution, and the mixture was shaken vigorously added with 4 mL of chloroform. The extracts were collected in a 10-milliliter flask. The

Preparation of Extracts
The seaweed and seagrass extracts were made by adding 5 g of dried seaweed or seagrass powder into 50 mL of three different solvents-chloroform, ethanol and distilled water-in a conical flask and placing the solution in a dark bottle in light agitation (AGIMATIC-N, J.P. SELECTA S. A, Barcelona, Spain) for 7 days. After that, the extracts were filtered through Whatman No. 1 filter papers and sterile cotton wools, and the supernatants were stored at 4 • C for future use [23,24].

Determination of Alkaloids
The alkaloid contents of K. alvarezii and C. serrulata were determined via the method proposed by Hikino et al. [25]. Next, 1 mL of test extract phosphate buffer (5 mL, pH 4.7) was added to 5 mL of bromocresol green solution, and the mixture was shaken vigorously added with 4 mL of chloroform. The extracts were collected in a 10-milliliter flask. The absorbance of the complex in chloroform was measured at 470 nm using a UV-Vis spectrophotometer (Shimadzu, Kyoto, Japan) against a prepared blank, as described above but without extract. Atropine (Sigma-Aldrich, St. Louis, MO, USA) was used as the standard, and the assay was compared to atropine equivalents.

Determination of Flavonoids
Total flavonoid content was determined via the aluminum chloride method [26] and using catechin (Sigma-Aldrich) as standard. Next, 1 mL of test sample and 4 mL of water were added to a volumetric flask (10-milliliter volume). After 5 min, 0.3 mL of 5% sodium nitrite and 0.3 mL of 10% aluminum chloride (Sisco Research Laboratories, Mumbai, India) were added. After 6 min of incubation at room temperature, 2 mL of 1-molarity sodium hydroxide (Sisco Research Laboratories) was added to the mixture. Afterwards, the final volume was brought to 10 mL via addition of distilled water. The absorbance of the reaction mixture was spectrophotometrically measured at 510 nm against a blank using a UV-Vis spectrophotometer (Shimadzu). The results obtained were expressed as catechin equivalents (mg catechin/g dried extract).

Determination of Tannins
The total tannin content extracts were determined according to the Julkunen-Titto [27] method. Firstly, 50-microliter extracts were mixed with 1.5 mL of 40% vanillin (Sisco Research Laboratories) (prepared with methanol), and 750 µL of HCl was then added. The solution was shaken vigorously and left at room temperature for 20 min in darkness. The absorbance of the mixtures was measured at 500 nm using a spectrophotometer (Shimadzu). A calibration curve was constructed using catechin (Sigma-Aldrich) in the range of 20-200 mg/L.

Determination of Phenolic Compounds
The total phenolic content in solvent extracts was determined using Folin-Ciocalteu's reagent, as proposed by Sangeeta and Vrunda [28]. During the procedure, different concentrations of the extracts were mixed with 0.4 mL of Folin-Ciocalteu's reagent (Sigma-Aldrich) (diluted 1:10 v/v). After 5 min, 4 mL of a sodium carbonate solution was added. The final volume of the tubes was brought to 10 mL by adding distilled water and left for 90 min at room temperature. The absorbance of the samples was measured against a blank sample at 750 nm using a spectrophotometer (Shimadzu). A calibration curve was constructed using 1,2-dihydroxybenzen (catechol) (Sigma-Aldrich) solutions as standards, and the total phenolic content of the extract was expressed in terms of the mg of catechol per g of dry weight.

Determination of Cardiac Glycosides
The cardiac glycoside content was determined using Buljet's reagent based on the method reported by El-Olemy et al. [29]. Firstly, 1 g of the fine powder of K. alvarezii and C. serrulata was soaked in 10 mL of 70% MeOH for 2 h and filtered. The extract obtained was then purified using lead acetate and Na 2 HPO 4 solution before the addition of freshly prepared Buljet's reagent (containing 95 mL of aqueous Picric acid and 5 mL of 10% aqueous NaOH) (Sigma-Aldrich). The difference between the intensity of colors of the experiment and blank samples gives an absorbance of 217 nm using a spectrophotometer (Shimadzu), which was used to calculate the concentration of glycosides.

Determination of Steroids
The steroid content was determined by Ejikeme et al. [26]. Firstly, 1 mL of test extract of the steroid solution was transferred into 10-milliliter volumetric flasks. Sulfuric acid (Sisco Research Laboratories) (4 N, 2 mL) and iron (III) chloride (Sisco Research Laboratories) (0.5% w/v, 2 mL) were added, followed by potassium hexacyanoferrate (III) solution) (Sisco Research Laboratories) (0.5% w/v, 0.5 mL). The mixture was heated via a Memmert WTB water bath (Memmert, Schutzart, Germany) that was maintained at 70 ± 20 • C for 30 min, along with shaking, and afterwards diluted to the mark with distilled water. The absorbance was measured using a 780 nm spectrophotometer (Shimadzu) against the reagent blank.

Determination of Carbohydrates
Carbohydrate content was estimated based on the phenol-sulfuric acid method [30]. In brief, 200 mg of a powdered sample, which was weighed using an analytical balance (OHAUS GA200, Nänikon Switzerland), was hydrolyzed by adding 5 mL of 2.5 N HCl. The sample was kept in boiling water, and after 3 h, the solution was neutralized with solid Na 2 CO 3 until effervescence ceased. The solution was made of up to 50 mL and centrifuged at 8000 rpm for 10 min in a centrifuge (Remi Lab World, Mumbai, India). Afterwards, the supernatant was aliquoted and brought up to 1 mL using deionized water, to which component 1 mL of phenol and 5 mL of 96% sulfuric acid (Sisco Research Laboratories) were previously added. After mixing the solution, it was kept in a water bath at 25 ± 1 • C for 20 min. The absorbance was measured at 490 nm using a UV-Vis spectrophotometer (Shimadzu) against the reagent blank.

Ash Content
The ash content was determined using the method of Yemm and Willis [30]. Firstly, 2 g of each sample was taken and weighed accurately using a Cobos CB balance (Barcelona, Spain) in a clean silica dish. The dish was first heated over a low burner flame. Next the dish was transferred to a SNOL muffle furnace (Utena, Lithuania) maintained at 500-550 • C for 3-5 h. The ash residue obtained was then cooled in a desiccator and weighed on a balance. The percentage of total ash content was calculated via the following formula: Total Ash Percent of plant sample (%) = [Weight of dry ash residue (g) ÷ Weight of plant sample (g)] × 100

Hydrogen Peroxide Radical Scavenging Activity
The antioxidant activity of seaweed and seagrass extracts was evaluated based on the hydrogen peroxide radical scavenging activity, as described by Ebrahimzadeh et al. [31]. The seaweed and seagrass extracts (100 µg/mL) were reacted with 0.6 mL of 40 mM H 2 O 2 solution prepared in phosphate buffer (pH 7.4) (Sisco Research Laboratories). After incubation at 37 • C for 10 min, absorbance was measured at 230 nm using a UV-Vis spectrophotometer (Shimadzu). Phosphate buffer was used as the corresponding blank solution. A similar procedure was repeated using distilled water instead of the extract, which served as a control. Ascorbic acid (Sigma-Aldrich) (20-100 µg/mL) was used as a standard.

In Vitro Antibacterial Activity of Seaweed and Seagrass against Human Pathogenic Bacteria
The antibacterial activity of seaweed and seagrass extracts was evaluated via the well diffusion method using a Muller-Hinton agar (Hi-Media, Mumbai, India). Approximately 100 µL of 10 5 CFU/mL of diluted inoculum of bacterial culture was applied to the surface of Muller-Hinton agar plates. The Muller-Hinton agar well was made using a well borer under aseptic conditions and filled with K. alvarezii and C. serrulata extracts, and methanol served as a positive control. The plates were incubated at 37 • C for bacterial growth, and the antibacterial activity of the seaweed and seagrass samples was evaluated by measuring the zone of inhibition (mm) in relation to the tested pathogenic bacteria. All experiments were performed in triplicate, and the data are expressed as the mean values of the experiments.

Characterization of the Active Compound by Gas Chromatography-Mass Spectrometry (GC-MS)
The crude extracts of K. alvarezii and C. serrulata were loaded into a silica gel (Hi-Media) packed column (20 cm length and 2 cm diameter) and eluted using n-hexane: ethyl acetate (50:50 v/v) (Sigma-Aldrich). The fractions were analyzed via a gas chromatograph GC-2010 interfaced with a quadrupole mass spectrometer QP-2010 (Shimadzu, Japan) analyzer, which used an Rtx-PCB capillary column (60 m × 0.25 mm i.d., 0.25 mm film thickness, Resteck, Bellefonte, PA, USA). Helium with a purity of 99.99% was used as the carrier gas at a flow rate of 1 mL/min. Next, 1 mL of extract was injected in spilt mode using an autosampler (Shimadzu). The injector port, interface and ion source temperature were set at 250, 270 and 230 • C, respectively. The mass spectrometer was operated in electron ionization (EI) mode at 70 eV and at an emission current of 60 mA. Full scan data were obtained in a mass range of 50-500 m/z. Interpretation of mass spectrum data was performed using the National Institute Standard and Technology (NIST) database.

Statistical Analysis
All determinations were given in terms of the mean ± standard deviation (SD). The results obtained were compared via one-way analysis of variance (ANOVA). The significance of the difference between means was determined via Duncan's multiple range test (p < 0.05) using SPPS version 14 (Chicago, IL, USA).

Phytochemical Analysis
Phytochemical analysis of K. alvarezii and C. serrulata revealed the presence of alkaloids (only in the case of K. alverazii), flavonoids, tannins, phenolic compounds, glycosides, steroids, carbohydrates and ashes. Among the six phytochemicals present in K. alvarezii, higher contents were found for phenolic compounds (3.39 ± 0.41 mg/g) and tannins (2.94 ± 0.41 mg/g). Both phenolic compounds and tannins have important roles as bioactive compounds. Phenols have important antioxidant, antimicrobial, anti-inflammatory and anticancer activities [32,33], whereas tannins are reported to have antiviral, antibacterial and antioxidant activities [34,35]. Among the five phytochemicals present in C. serrulata, the highest contents were found for glycosides (2.47 ± 0.41 mg/g) and flavonoids (2.11 ± 1.40 mg/g) ( Table 1). With respect to glycosides, it was reported that they have antioxidants and anti-inflammatory activities, which find application in the prevention and managements of several human diseases [34]. Flavonoids also make up an important phytochemical group due to their antimicrobial, antiviral, antioxidant and spasmolytic activities [35]. These constituents significantly contribute to the biological activity of seaweeds and seagrass [36]. Similar observations were also made by other works [37,38], which found tannins, flavonoids, phenolic compounds, carotenoids and polysaccharides in both seaweed and seagrasses. In the present study, K. alvarezii showed a higher tannin content (2.94 ± 0.41 mg catechin equivalent (CAE)/g) than C. serrulata (1.94± 0.85 mg CAE/g). Similarly, Deyad and Ward [35] reported similar tannin content in the brown seaweed Dictyota dichotoma (2.12 ± 0.45 mg CAE/g), whereas Domettila et al. [32] reported a higher presence of tannins in the red seaweed Sargassum wightii (27.54 ± 0.54 mg CAE/g). In previous studies [9], the presence of tannins in C. serrulata (264.71 mg/mL tannic acid equivalence) was reported. Similarly, another work reported the presence of tannins in the seagrass Syringodium isoetifolium (80.65 ± 5.64 mg CAE/g [39]. Tannins are polyphenols, which have a large influence on the nutritive value of humans and animals due to their antimicrobial, anti-inflammatory, and astringent activities [9]. Flavonoid content was similar in both K. alvarezii and C. serrulata, although in global terms, it was found in lower amounts than in previous works. Vaghela et al. [40] found a much higher flavonoid content (15.26 ± 0.95 mg CAE 100 g −1 ). Similarly, Smadi et al. [41] reported the flavonoid content of C. nodosa to be 3.98 ± 0.03 mg CAE/g, which is comparatively higher than the results of the present study.
The phenolic compound content in seaweeds is, in part, responsible for their scavenging activity, which protects them against lipid oxidation [43]. In this work, K. alvarezii showed a higher phenolic content (3.39 ± 0.45 mg gallic acid equivalents (GAE)/g) than C. serrulata (1.01 ± 0.39 mg GAE/g). Previously, other authors reported a significantly higher content of phenolic compounds in both K. alvarezii (3.14 ± 0.14 mg GAE/g) [44] and Kappaphycus striatum (7.24 ± 0.21 mg GAE/g) [45]. Regarding C. serrulata, the results obtained in the current work were also significantly lower than those reported by Libin et al. [17] for C. serrulata (2.98 ± 0.12 mg GAE/g) and Cynodocea rotundata (2.04 ± 0.1) [46]. The phenolic contents of seaweed and seagrass depend on the solvent used to analyze the extraction process, environment, habitat and biomass.
With respect to carbohydrates and ash content, the carbohydrate content of K. alvarezii was 2.57 ± 1.89 mg/g DW, while that of C. serrulata was 1.44 ± 1.75 mg/g DW. The wide variation in the carbohydrate content observed between seaweed and seagrass might be due to the influence of different factors, such as salinity, temperature and sunlight intensity. Regarding ash, K. alvarezii had a higher ash content (8.5 ± 0.95 g/100 g) than C. serrulata (6.9 ± 0.49 g/100 g). High ash content showed the presence of appreciable amounts of diverse minerals found in both seaweed and seagrass.

Antioxidant Activity
Antioxidant effectiveness is measured by monitoring the inhibition of oxidation of a suitable substrate [15]. In biological systems, antioxidant effectiveness is classified into two groups: evaluation of lipid peroxidation and measurement of free radical scavenging ability [31]. The in vitro antioxidant activiies of K. alvarezii and C. serrulata extracts were evaluated based on hydrogen peroxide radical scavenging activity, and K. alvarezii had higher scavenging activity (27.9 ± 0.1%) than C. serrulata (22.1 ± 0.1%). Regarding K. alvarezii, the results obtained were higher than those previously reported by other authors, such as Farah et al. [37] or Chew et al. [54], who reported lower (18.34 ± 0.57% and 11.8 ± 5.7%, respectively) 2,2-Diphenyl-1-picrylhydrazyl (DPPH) scavenging activity. Regarding C. serrulata, the DPPH scavenging activity results obtained were lower than those obtained by Kannan et al. [50] (61.85 ± 0.95%) regarding free radical scavenging activity from the same seagrass species, though higher than those results reported by Rengasamy et al., [33] (6.65 ± 0.12%) for other Cymodocea species, such as C. rotundata.

Antimicrobial Activity
The antibacterial activity of both K. alvarezii and C. serrulata were investigated using chloroform extracts based on those reported by Pusparaj et al. [14], who reported that the best inhibitory effects of K. alvarezii were reported using chloroform extracts. The antibacterial activities of both K. alvarezii and C. serrulata depend on the presence of bioactive compounds, phenolic content and free radical scavenging activity [55]. In all cases, inhibitory activities against the five pathogenic bacteria investigated were detected ( Table 2). The higher inhibitory activity was observed in K. alvarezii (26 ± 0.03 mm) against Bacillus subtilis, as well as in the case of C. serrulate, which exhibited maximum inhibitory activity (26 ± 0.08 mm) against Vibrio parahaemolyticus. The chloroform extract of K. alvarezii showed maximum activity of 26 ± 0.03 mm against B. subtilis at 100 µg/mL, and C. serrulata showed maximum activity of 26 ± 0.08 mm against V. parahaemolyticus at 100 µg/mL and minimum activity of 22 ± 0.01 mm and 20 ± 0.04 mm against Vibrio alginolyticus at 100 µg/mL in both K. alvarezii and C. serrulata, respectively (Table 2). Jaswir et al. [55] reported maximum inhibitory activity (12 ± 1.02 mm) against B. subtilis using the methanolic extract of K. alvarezii. Similarly, Pusparaj et al. [14] reported the antibacterial activity of K. alvarezii against six human pathogens: Staphylococcus aureus, B. subtilis, Lactobacillus acidophilus, Pseudomonas aeruginosa, Escherichia coli and Proteus mirabillis. He also reported that the best activity was recorded in chloroform extracts. Kumar et al. [56] studied the antibacterial activity of C. serrulata against four fish-borne pathogens, namely Bacillus cereus, B. subtilis, E. coli and Micrococcus luteus, and reported that C. serrulata was effective against several Bacillus species.

Conclusions
The red seaweed K. alvarezii and seagrass C. serrulata examined in the present study were found to possess rich sources of phytochemicals. The antioxidant properties of both seaweed and seagrass reveal that they have appreciable levels of protection against free radicals.